Integral membrane protein | EPFL Graph Search

An integral, or intrinsic, membrane protein (IMP) is a type of membrane protein that is permanently attached to the biological membrane. All transmembrane proteins are IMPs, but not all IMPs are transmembrane proteins. IMPs comprise a significant fraction of the proteins encoded in an organism's genome. Proteins that cross the membrane are surrounded by annular lipids, which are defined as lipids that are in direct contact with a membrane protein. Such proteins can only be separated from the membranes by using detergents, nonpolar solvents, or sometimes denaturing agents. Structure Three-dimensional structures of ~160 different integral membrane proteins have been determined at atomic resolution by X-ray crystallography or nuclear magnetic resonance spectroscopy. They are challenging subjects for study owing to the difficulties associated with extraction and crystallization. In addition, structures of many water-soluble protein domains of IMPs are available in the Protein D

Proteins interacting with PfEMP1 in P. falciparum infected erythrocyte

Damien Jacot

During infection in humans, P. falciparum invades and refurbishes the red blood cells (RBCs) in order to persist and proliferate. Parasite survival depends on expression of a parasite encoded cytoadherence ligand at the surface of the infected RBC called PfEMP1 (P. falciparum erythrocyte membrane protein 1). PfEMP1 comprises a N-terminal extracellular part, which mediates binding to receptors at the host endothelium. It also contains a C-terminal part which is conserved among all PfEMP1 molecules. This acidic terminal segment (ATS) anchors PfEMP1 to the membrane of infected RBCs and has a role for trafficking. To bring proteins to the RBC surface, P. falciparum builds its own trafficking machinery de novo since the erythrocytes do not contain secretory organelles. These new structures include parasite derived compartments called Maurer's clefts (MCs) located at the RBC periphery. The resident MCs protein MAHRP1 (membrane-associated histidine-rich protein 1) has been shown to be critical for PfEMP1 trafficking. A MAHRP1 KO strain had no PfEMP1 on the RBC surface but accumulated PfEMP1 within the parasite. MAHRP1 is a 28.9kDa protein with 249 amino acids containing a N-terminal, a transmembrane, and a C-terminal domain comprising histidine-rich repeats. To re-establish PfEMP1 trafficking, the MAHRP1 KO parasite line was complemented with various truncated HA-tagged fragments of MAHRP1. Immunofluorescent assays (IFAs) showed that the Cterminal domain with the histidine-rich repeats was not essential for PfEMP1 trafficking.

{MAHRP1}_{1-130}

and

{MAHRP1}_{1-169}

were exported to MCs and restored trafficking of PfEMP1. Shorter MAHRP1 fragments were not exported to MCs and PfEMP1 transport remained impaired. New transfection constructs were designed including the C-terminal part to identify important domain(s) of MAHRP1 for PfEMP1 trafficking. To identify other proteins interacting with PfEMP1, the ATS domain of PfEMP1 was recombinantly expressed and used in pull-down experiments. In these experiments using parasite lysates, PF14_0377 was identified as a potential interaction partner. This protein has been described as putative, vesicular-associated membrane protein. To subsequently assess the interaction(s) and the localization(s), PF14_0377 was cloned and transfected in 3D7 for IFAs. Due to time restrictions all these new transfectants could not be analyzed completely

2009

Investigating molecular interactions of G protein coupled receptors by fluorescence techniques

Jean-Baptiste Perez

G Protein coupled receptors (GPCRs) constitute an abundant family of membrane proteins which play a central role in cellular signaling and are important targets for modern medicine. Despite intensive research, central questions of the molecular mechanism of GPCR mediated signal transduction remain unresolved. This thesis concerns α1b-adrenergic receptor (α1b-AR) as a prototypic GPCR. The question whether ARs form homo- or hetero-oligomers was investigated using fluorescence resonance energy transfer (FRET). An important finding was that both α1a and α1b-AR subtypes form homo-oligomers. Also hetero-oligomers have been observed between the α1b- and the α1a-AR subtypes, but not with less related receptors. Another interesting finding concerns α1-AR co-internalization which correlates with the ability of the receptor to hetero-oligomerize. Investigating particular processes in living cells is often complicated by the complex cellular environment. Here we have developed a rather simple approach to study transmembrane signaling by using supported cell membrane sheets. They were prepared by pressing a glass coverslip on the apical part of cultured cells, ripping off large regions of the native plasma membrane. For the α1b-AR we still could observe ligand binding to such sheets, indicating that GPCR functionality was at least partly conserved. Due to the absence of cytosolic autofluorescence of the cells, supported membrane sheets were also found to be suited for single-molecule microscopy. Because both leaflets of the supported membranes remained fluid, it was possible to follow the diffusion of certain membrane proteins. For example using fluorescence recovery after photobleaching (FRAP) and single-molecule microscopy, we investigated how G proteins diffused along the membrane and compartmentalized. The predominant role of lipid anchors in G protein localization was highlighted by measuring the diffusion of two Gαq proteins fused with citrine at two different positions and citrine based constructs designed to mimic the monomeric and the trimeric form of a G protein. Finally, first results were obtained showing the feasibility to form supported cell membrane sheets on microstructured devices consisting of a planar silicon support perforated with arrays of holes. This geometry provides a full accessibility to both intra- and extracellular sides of the bilayer. This concept could potentially be applied for the development of chip-based screening assays.

EPFL2005